Functional Analysis Of Three ECF-σ Factors σ₂₅, σ₅and σ₁₆ In Streptomyces Avermitilis

Extracytoplasmic function (ECF) σ factors represent an abundant fundamental principle of bacterial signal transduction and play key roles in redirecting RNA polymerase to initiate transcription from alternative promoters for adaptation to extracellular signals and stress response. However, the functions of ECF σ factors in antibiotic biosynthesis remain poorly understood.Avermectins, produced by Streptomyces avermitilis, are major commercial antiparasitic agents widely utilized in agriculture and animal husbandry. We previously performed comparative transcriptome analysis between S. avermitilis wild-type strain ATCC31267and the avermectin-overproducing strain76-02-e using agilent expression microarray to reveal novel regulators that influence avermectin biosynthesis. Analysis of microarray data revealed three differentially expressed ECF-σ factor genes sig25, sig5and sigl6. In the present study, the roles of ECF σ factors σ, σ5and σ16in antibiotic biosynthesis, morphological differentiation and stress response were investigated.sig25(SAV3351) encodes a putative ECF-σ25. Fermentation and morphological observation of sig25gene deletion, complementation and overexpression strains showed that a25has no effect on growth or morphology, but inhibits avermectin production and promotes oligomycin production. RT-qPCR, EMS A and in vitro transcription assays showed that σ25indirectly inhibits avermectin production by affecting the transcription of the pathway-specific activator gene aveR, whereas it directly activates oligomycin production by initiating transcription of the pathway-specific activator gene olmRI. a25was also found to initiate its own transcription. Stress tests suggested that a25is involved in responding to hot shock, SDS treatment, extreme pH and osmotic stress. The divergently transcribed genes smrAB are located upstream of sig25and encode a putative two-component system (TCS). The response regulator SmrA directly activates sig25transcription by binding to direct repeat sequences CTGTGA-n5-CTGTGA. SmrAB had the similar effect on growth, morphology, avermectin and oligomycin production to a25. These findings indicated that a25and SmrAB function similarly in the regulation of antibiotic production. Blast analysis revealed that the promoter region of SAV111(encoding a LuxR family transcriptional regulator) is very similar to that of sig25. SAV111was proven to be a target gene of SmrA and a25, and SAV111was shown to directly promote avermectin biosynthesis by binding to the promoter region of structural gene aveAl. The conserved SmrA binding site was used to predict putative target genes of SmrA and experiments were performed to investigate whether these genes are also target genes of σ25. RT-qPCR and EMSA assays showed that SmrA and σ25can co-regulate the expression of SAV2630(whiG), SAV3434, SAV4621, SAV5010and SAV6537, which are involved in morphological differentiation, DNA replication, and primary metabolism. SmrA and σ25can also co-regulate the expression of amtBl, gdhAl and glnA2, which participate in nitrogen metabolism. In addition, SmrA and σ25both have respective target genes.sig5(SAV614) and sigl6(SAV1195) encode ECF-σ factor σ5and σ16, respectively. Determination of avermectin production and morphological observation in gene deletion, complementation and overexpression strains implied that σ5and σ16both inhibit avermectin biosynthesis, but have no effect on growth or morphology. Further experiments indicated that the inhibitory role of σ5and σ16in avermectin production is indirect. RT-qPCR and ChIP assays showed that σ5positively regulated the transcription of itself and adjacent genes by binding to the promoter regions of sig5, SAV612, SAV615and SAV618. Experiments should be conducted to investigate whether these target genes of σ5affect avermectin production. Stress tests suggested that σ5is involved in responding to osmotic stress. EMSA assays showed that σ16can not bind to the promoter of operon sigl6-SAV1196, but bind to the promoter of branched-chain amino acid ABC transporter operon SAV1190-SAV1194. Branched-chain amino acids provide precursors for avermectin biosynthesis. The relationship of branched-chain amino acid ABC transporter system SAV1190-SAV1194with avermectin biosynthesis should be investigated further.